In addition to isolation, the analysis of oligonucleotides by LC/MS presents unique challenges compared with the study of small molecule therapeutics. Being reasonably long anionic polymers, oligonucleotides are not generally retained by reversed-phase HPLC and require the use of ion-pairing buffers to obtain retention for analysis by LC/MS.
A good review of the balance between ion-paring mediated retention and ion-suppression is covered in detail in a May 1, 2009 article in GEN (available online). However separation of the oligonucleotide from matrix interference is an additional requirement.
An example of isolating an oligonucleotide from LC/MS-interfering matrix contaminants is shown in Figure 2. In this example a 19 mer phosphorothioate oligonucleotide was extracted from plasma using the Clarity OTX protocol and analyzed on an Oligo HTCS LC/MS system (Novatia) utilizing a Clarity Oligo-MS column.
Figure 2A shows the LC/MS TIC chromatogram of the extracted oligonucleotide and demonstrates high recovery and removal of MS-interfering plasma matrix contaminants that might co-migrate on LC. (A few matrix peaks are observed that elute away from the oligonucleotide peak.) The isolated MS spectra of the 19 mer oligonucleotide peak at 14.3 minutes retention time is shown in Figure 2B. Major ions corresponding to the -6, -7, and -8 ions of the parent oligonucleotide are readily seen.
Using an electrospray ionization mass spectrometer, oligonucleotides generally are observed as a mixture of negatively charged ions that do not readily translate to a specific parent mass of the oligonucleotide. This data is further complicated when multiple components co-migrate by LC.
To identify all of the components in an oligonucleotide mixture, deconvolution software is needed to translate signal contributions from all the different ions to the parent mass of the expected oligonucleotide as well as any major contaminants. The raw spectra from Figure 2B were input into the Novatia ProMass deconvolution software to generate the reconstructed mass spectra shown in Figure 2C.
Note that processed ions generate a mass spectrum that corresponds to the expected mass of the full-length 19 mer oligonucleotide. Also note minor components that correspond to low-level sodium adducts of the full-length oligonucleotide as well as a low-level peak corresponding to the expected mass of a depurinated oligonucleotide. While that depurinated oligo peak might possibly be due to an MS artifact, this data demonstrates the necessity of having deconvolution software for analyzing oligonucleotide samples to quantitate the oligonucleotide as well as any low-level metabolites.
In the drug-development pipeline for oligonucleotide therapeutics, the most recent challenge has been developing robust, high-throughput methods for preparing and analyzing ADME/pharmacokinetic samples. The results shown here demonstrate the suitability of moving to SPE-only protocols using Clarity OTX cartridges as a way of bypassing the manually intensive LLE steps and automating the sample-preparation process.
Multiplexing the process in a 96-well plate format offers the prospect of further increasing throughput. Equally important in obtaining useful LC/MS data for oligonucleotides is using proper chromatography conditions on a high-resolution HPLC column coupled to a sensitive MS system. Finally, having proper bioinformatics to reconstruct MS spectra is crucial to quantitating oligonucleotides and their metabolites from biological samples.